Process for producing xylitol by fermentation

ABSTRACT

A process for producing xylitol by fermentation (xylitol is useful as sweetening materials or treating agents of diabetics), which comprises (i) culturing yeasts, for example, Debaryomyces sake (ATCC 20,212) in a culture medium containing glucose so as to produce D-arabitol, (ii) after the sterilization of said culture medium, culturing acetic acid bacteria, for example, Acetobacter suboxydans ATCC 621 therein so as to produce Dxylulose, and further, (iii) with or without separating Dxylulose from said broth, culturing yeasts, for example, Candida guilliermondii var. soya ATCC 20,216 in a culture medium containing the D-xylulose as a sugar source so as to produce xylitol.

United States Patent Inventors lllroshi Onishi;

Toshiyukl Suzuki, both 01 Node-sh], Japan Appl. No. Filed PatentedAssignee 840,034 July 8, 1969 Nov. 9, 1971 Priorities Noda Institute forScientific Research Oct. 31, 1968, Japan, No. 43/78945 PROCESS FORPRODUCING XYLITOL BY FERMENTATION 7 Claims, No Drawings US. Cl 195/37,195/43 Int. Cl Cl2c 1/00 Field of Search 195/37, 42, 1 1 1, 43

References Cited UNITED STATES PATENTS 5/1961 Onishi 195/37 AssistantExaminerGary M. Nath Attorney-Cushman, Darby & Cushman ABSTRACT: Aprocess for producing xylitol by fermentation (xylitol is useful assweetening materials or treating agents of diabetics), which comprises(i) culturing yeasts, for example, Debaryomyces sake (ATCC 20,2 l 2) ina culture medium containing glucose so as to produce D-arabitol, (ii)after the sterilization of said culture medium, culturing acetic acidbacteria, for example, Acetobacter suboxydans ATCC 621 therein so as toproduce D-xylulose, and further, (iii) with or without separatingD-xylulose from said broth, culturing yeasts, for example, Candidaguilliermondii var. soya ATCC 20,216 in a culture medium containing theD-xylulose as a sugar source so as to produce xylitol.

PROCESS FOR PRODUCING XYLI'POL BY FERMENTATION BACKGROUND OF THEINVENTION Field of the Invention Description of Prior Art Xylitol hasbeen used as sweetening materials or medicines for treatment ofdiabetes. Xylitol is produced by the reduction of D-xylose. However,economical conversion and recovery of xylose has not yet been effectedso that xylitol cannot be considered a commercially available polyol.(Encyclopedia of Chemical Technology. the lnterscience Encyclopedia Inc.New York, vol. 1, p. 323). D-xylose is expensive, and accordingly,xylitol is also very high-priced.

The object of the present invention is to provide a process forproducing xylitol from D-xylulose by fermentation. D- xylulose which isa starting material of the objective substance is easily produced fromglucose by fermentation successively. Accordingly, the present inventionis economically valuable because xylitol is obtained from glucose at lowcost.

In order to obtain D-xylulose from glucose, D-arabitol is first producedfrom glucose and subsequently, the resulting arabitol is converted intoD-xylulose. The former procedure (glucose- D-arabitol) is well knownsee, for example, CA. 59, p. 3288f(1963) and CA. 59, p. 1054b (1963).Namely, it comprises inoculating Pichia miso (ATCC 20,210), Torulopsisfamata (ATCC 20,214) and Candida polymorpha (ATCC 20,213) in a culturemedium containing glucose as a main sugar source and conducting thecultivation under aerobic conditions. Further, the latter procedure(D-arabitobD-xylulose) is also well known and it depends upon the actionof Acerobacter suboxydans (ATCC 621). However, these wellknown methodsare conducted in a single step and no method for conducting themsuccessively has been reported. The successive method is very convenientbecause a yeast growing in former procedure is sterilized with heatingso as to obtain a yeast extract and the resulting yeast extract is usedas a nutrirional source of acetic acid bacteria in the latter procedure.

SUMMARY OF THE INVENTION The present invention relates to a process forproducing xylitol from glucose by fermentation which comprisesinoculating yeasts capable of producing D-arabitol from glucose in aculture medium containing glucose as a main sugar source, conductingculturing under aerobic conditions so as to produce and accumulateD-arabitol, subsequently inoculating acetic acid bacteria into saidmedium so as to oxidize and ferment resulting D-arabitol, thereby,producing D-xylulose. Moreover the present invention also relates toinoculating yeasts capable of producing xylitol from D-xylulose in a culture medium containing the D-xylulose as a sugar source, conductingcultivation and fermentation thereof under aerobic conditions andconsequently, obtaining xylitol. lf commercially available, D-xylulosecan be used to produce xylitol according to the above-mentioned process.The following discussion demonstrates the process of this inventionwherein commercially available glucose is utilized as the startingmaterial.

1. Production of D-arabitol from glucose A process for producingD-arabitol in high yield by aerobic fermentation of yeasts by use ofglucose as a main sugar source has already been clarified by one of thepresent inventors. C.A. 59, p. 3288f( 1963) and CA. 59, p. 1054):(1963). In carrying out a process according to the invention of thepresent application. any yeasts which have an ability to produceD-arabitol from glucose may be used. For example,

Pichra miro 3362 (ATCC 20,210) and Pichia membranaefacr'ensbelonging togenus Pichia, Hansenula miso B (ATCC 20,21 1 and Hansenula satumusbelonging to genus HanLrenu- Ia, Debaryomyces sake (ATCC 20,212) andDebaryomycer, miso var. 1 belonging to genus Debaryomyces, Sacchammycesrouxii, (ATCC 13,356) and Saccharomyces, acidifaciens belonging to genusSaccharomyces, Candida polymorpha 3544 (ATCC 20,213) and Candida cruseibelonging to genus Candida and other yeasts such as Tamlopris famara3555 (ATCC 20,214), Torulopsis osloensrlr, Rhodotorula rosa, andfurther, the yeasts demonstrated in Hiroshi Onishi: Bull. Agr. Chem.Soc. Japan 24, 131 (1960) may be used.

In producing D-arabitol from glucose by use of these yeasts, theabove-mentioned strains are precultured in an appropriate medium for thegrowth of micro-organisms, for example, a culture medium forfermentation, are inoculated into a culture medium containing glucose asa main sugar source and incubated. The sugar concentration can be variedin a wide range, preferably 10-30 percent. Other nutrients necessary forthe growth of yeasts are added to a culture medium. As for nitrogenouscompounds, organic substances such as casein hydrolyzate, peptone, aminoacids, corn steep liquor, urea and the like, or inorganic substancessuch as ammonium sulfate, ammonium chloride, and the like are usable.Moreover, inorganic substances such as phosphates, magnesium salts, andcalcium salts, yeast extract, vitamins and the like may be used.

The fermentation is conducted under aerobic conditions while maintainingthe culture medium in which the abovementioned strains have beeninoculated at a temperature of about 30 C. After 2-10 days, the sugar isconsumed and D- arabitol is produced and accumulated in the culturemedium.

For example, in case of culturing in the sugar concentration of 30percent by weight of glucose, the yield of arabitol based on sugar is asfollows.

Pichia miso 3362 (ATCC 20,210) 29% Hansenula miso B lFO-l 47(ATCC20.2I1) 21% Debaryomyces sake lFO-0060 (ATCC 20,212) 35%Saccharomyces rouxii (ATCC l3,356) 2291: Candida polymorpha 3554 (ATCC20.2 I 3) 37% Torulopsis famata 3555 (ATCC 20.214) 51% 11 Production ofD-xylulose from D-arabitol The fermentation liquor containing D-arabitolas obtained above is adjusted to about pH 6.0 and is then sterilizedwith heating. The cell bodies of yeasts are killed and simultaneously,the component thereof is eluted in the broth. To this broth acetic acidbacteria, for example, Acetobacter suboxydans ATCC-62l or Gluconobacterroseus H [AM 1840 and the like are inoculated and fermented aerobicallyat about 30 C. As a result, D-xylulose is produced in high yield by theoxidation of D-arabitol. The adjustment of the pH mentioned above isconducted by use of caustic soda. The extract from yeasts employed inthe proceeding procedure (1) is nutritious and therefor, the brothobtained in the procedure (1) can be used as the culture medium of thepresent procedure as it is. As delivered above, D-xylulose can beproduced from glucose according to a simple method by culturing yeastsand acetic acid bacteria successively, without conducting suchcomplicated procedures as purification and so on.

lll Production of xylitol from D-xylulose Corn steep liquor is suppliedto the fermentation liquor obtained in the above-mentioned procedure(11) as a nutritional source and this fennentation liquor is adjusted topH 6.0 and sterilized. The fermentation is conducted by culturing yeastscapable of converting D-Xylulose into xylitol under aerobic conditions.

After cell bodies of nitrogenous substances are removed from afermentation liquor containing D-xylulose obtained by the procedure(ll), the fermentation liquor is concentrated under reduced pressure soas to obtain a dried substance, and the resulting dried substance istreated by the extraction of hot ethanol to separate D-xylulose. Theabove-mentioned microorganisms may be cultured in a culture medium (pHabout 6.0) containing the thus-isolated D-xylulose as a sugar source andother nutritional sources as mentioned above according to theconventional method to produce xylitol.

As appropriate yeasts in the present procedure, Candida tmpicalis 3527ATCC 20,215, Candida arborea ATCC Candida guilliermondii var. soya ATCC20,216, Pichia farinosa ATCC 20,218, Hansenula suaveolens ATCC 20,219,Monilia vini ATCC 20,217, Saccharomyces rowrii ATCC 133,561,Zygosaccharomyces polymorphus, Zygosaccharomycer gracilis var. italicusATCC 2623 and Debaryomyces hansenii ATCC 20,220 are used.

In producing xylitol from D-xylulose by use of these yeasts, theabove-mentioned strains precultured in a suitable medium in which saidstrains are capable of growing, such as a culture medium forfermentation, are inoculated in a culture medium containing D-xyluloseas a main sugar source and incubated. In this case, the concentration ofD-xylulose is suitably -10 percent in general. Other essential nutrientsnecessary for the growth of yeasts are added to the culture medium. Asfor nitrogen compounds, organic substances such as corn steep liquor,casein hydrolyzate, peptone, amino acids, urea and the like or inorganicsubstances such as ammonium sulfate, ammonium chloride and so on may beused. Moreover, inorganic substances such as phosphates, magnesiumsalts, calcium salts and the like, yeast extract and vitamins can beused. Particularly, when the broth of the procedure (11) is used, it issufficient to add only corn steep liquor to the culture medium in aconcentration of 2-8 (w./v.) percent. The adjustment of pH is conductedby use of caustic soda.

Culturing is carried out at a temperature of about 30 C. under aerobicconditions. The fermentation is conducted for 3-6 days by conductingsuitable aeration or supplying air with a shaking method.

For example, in the case of conducting the cultivation in a culturemedium containing 5 (w./v.) percent of D-xylulose and casein hydrolyzateas a nitrogen source, the yield of xylitol based on sugar is as follows.

Candida tropicnlis ATCC 20,215 16.6% Candida guilliermondii var. soyuATCC 20,216 25.4% Moniliu vini ATCC 20,217 16.2% Pichia farinosa ATCC20,218 19.6% Hanscnula suaveolens ATCC 20.219 7.1% Snccharornyces rouxiiATCC 13,356 27.1% Zygosacchuromyces polymorphus 48.3% Zygosaccharomyccsgracllis var. italicus ATCC 2.623 45.8% Dcbaryomyces hansenii ATCC20,220 25.8%

As shown in example 1, in the case culturing Candida guilliermandii var.soya ATCC 20,216 in a culture medium containing com steep liquor as anitrogen source, xylitol is obtained in the high yield of 40 percentbased on sugar. The yield thereof has remarkably been elevated.

RECOVERY A clarified solution obtained by filtering cell bodies andinsoluble material from the fermentation liquor obtained by theprocedure (111) is concentrated under vacuum at a low temperature of 50C. or lyophilized to remove water. The dried materials is extracted withhot ethanol. in the case that the fermentation liquor containsD-xylulose which has not been fermented yet, it is treated with an ionexchange resin Amberlite 1RA-400 (manufactured'by the Organo Company) toobtain the fraction of xylitol. Seed xylitol crystal is added to theethanol extract and then the extract is allowed to stand andcrystallize. Recrystallization is conducted so as to obtain purecrystals. Thus obtained crystal has a melting point of 92.593.5 1 C. anda sweet taste. The molecular formula thereof is C l-1, 0 and it has nooptical activity. The result of the analysis of infrared absorptionspectrum is entirely identical to that of xylitol. Consequently, thiscrystal is identified with xylitol from the above-mentioned facts.

PREFERRED EMBODIMENT OF THE INVENTION Example 1 Debaryomyces sake ATCC20,212 was inoculated in a culture medium containing 15 (w./v.) percentglucose, 4.0 (w./v.) percent com steep liquor, 0.1 (w./v.) percent acidpotassium phosphate, 0.05 (w./v.) percent magnesium sulfate, 0.01(w./v.) percent calcium chloride, 0.0] (w./v.) percent sodium chlorideand balance being water at pH 6.0 and cultured with shaking at 30 C. for4 days. 5.34 g./ ml. of D- arabitol was produced and accumulatedconsuming sugar. Further, the culture medium was adjusted to pH 6 withcaustic soda and then autoclaved at C. for 15 minutes. ThereintoAcerobacter suboxydans ATCC 621 was inoculated and oxidatively fermentedat 30 C. for 2 days, thereby 5.00 g./100 ml. of D-xylulose was producedand accumulated. Next, 4.0 (w./v.) percent corn steep liquor was furtheradded to the fermentation liquor without isolating and recoveringD-xylulose from the fermentation liquor and the said fermentation liquorwas adjusted to pH 6.0 and sterilized at 1 10 C. for 5 minutes.Thereinto Candida guilliermondii var. soya ATCC 20,216 was inoculatedand cultured with shaking at 30 C. for 3 days. Consequently 2.00 g./ 100ml. of xylitol was produced completely consuming sugar. The yield ofxylitol from the initial glucose was 13.3 percent.

Example 2 Candida polymorpha ATCC 20,213 was inoculated into a culturemedium consisting of 10 (w./v.) percent glucose, 0.4 (w./v.) percentcasein hydrolyzate, 0.1 (w./v.) percent acid potassium phosphate, 0.05(w./v.) percent magnesium sulfate, 0.01 (w./v.) percent calciumchloride, 0.01 (w./v.) percent sodium chloride, 0.1 (w./v.) percentyeast extract and balance being water and cultured with shaking at 30 C.for 5 days. 4.5 g. of D-arabitol was obtained per 100 ml. of thefermentation liquor.

This fermentation liquor was adjusted to pH 6 with caustic soda andautoclaved at 120 C. for 15 minutes. Thereafter, Acetobacter suboxydansATCC 621 was inoculated into the said fermentation liquor and incubatedat 30 C. for 2 days. Consequently, 4.5 g./100 of D-xylulose (the yieldof D-xylulose from glucose was 45 percent based on sugar) was obtained.

The fermentation liquor was then separated by centrifugation so as toremove cell bodies and insoluble materials and further, nitrogenoussubstances was removed therefrom by the treatment of zinc sulfate.Moreover, the resulting fermentation liquor was concentrated to drynessunder reduced pressure, extracted with hot ethanol, treated with activecarbon to remove impurities and then, the fraction of D-xylulose wasobtained by evaporating alcohol under reduced pressure.

Candida guillumondii var. soya ATCC 20,216 was inoculated in a culturemedium consisting of 5 (w./v.) percent D- xylulose, 0.4 (w./v.) percentcasein hydrolyzate, 0.1 (w./v.) percent acid potassium phosphate, 0.05(w./v.) percent by weight of magnesium sulfate, 0.01 (w./v.) percentcalcium chloride, 0.01 (w./v.) percent sodium chloride, 0.1 (w./v.)percent yeast extract and balance being water, by use of the resultingD-xylulose fraction and cultured with shaking at 30 C. for 3 days.Consequently, 1.4 g. of xylitol was obtained per 100 ml. of thefermentation liquor (the yield of xylitol was 12.6 percent based onglucose and 28 percent based on xylulose). The clarified solutionobtained by filtering cell bodies and protein from said fermentationliquor was concentrated under vacuum or lyophilized so as to removemoisture. The dried material was extracted with hot ethanol. 1fD-xylulose remains unfermented, the broth was treated by an ion exchangeresin 1RA 400 (manufactured by the Organo Company) to remove the sugarbefore ethanol extraction. Seed xylitol crystal was added to thisextract and kept to stand to crystallize. Pure crystals were obtained byrepeating recrystallization.

In case of using respective strains of Candida tropicalis ATCC 20,215,Monilia vini ATCC 20,217, Pichia farinosa ATCC 20,218 and Hansenulasuavealens ATCC 20,219 instead of the above-mentioned Candidaguilliermandii var. soya ATCC 20,216, the result thereof are shown intable 1.

Example 3 Pichia miso ATCC 20,210 was inoculated in a culture mediumhaving the same composition as described in example 2 except that 25(w./v.) percent glucose was used and cultured with shaking at 30 C. fordays. 7.5 g./ 100 ml. of D-arabitol was obtained.

The similar oxidation fermentation as in example 2 was conducted in thisfermentation liquor using Acetobacter suboxydans ATCC 621 so as toobtain a D-xylulose fraction and then, it was prepared to be 5 percentconcentration and subsequently, the fermentation was conducted usingDebaryomyces Hansenii ATCC 20,220 at 30 C. for 4 days. 1.3 g. of xylitolwas obtained per 100 ml. of the fermentation liquor. The yield ofxylitol from glucose was 7.8 percent based on sugar.

Example 4 Torulopris famata ATCC 20,214 was inoculated into a culturemedium consisting of 10 (w./v.) percent glucose, 0.4 (w./v.) percentcasein hydrolyzate, 0.1 (w./v.) percent acid potassium phosphate, 0.05(w./v.) percent magnesium sulfate, 0.01 (w./v.) percent calciumchloride, 0.01 (w./v.) percent sodium chloride, 0.1 (w./v.) percentyeast extract and balance being water and cultured with shaking at 30 C.for 5 days. Consequently, 4.8 g./l00 ml. of D-arabitol was produced andaccumulated consuming sugar. After adjusting the fermentation liquor topH 6 with caustic soda, it was autoclaved at 120 C. for minutes.Thereinto Gluconobacter roseus H, was inoculated and after 2 daysoxidative fermentation at 30 C., 4 g./ l 00 ml. of D-xylulose wasproduced and accumulated.

The fraction of D-xylulose was obtained by the same treatment asdescribed in example 2. Candida tropicalis ATCC 20,215 was inoculatedinto a culture medium having the same composition as described inexample 2 except that 5 percent by weight of D-xylulose was used andcultured with shaking at 30 C. for 4 days. Consequently, 0.83 g. ofxylitol (the yield of xylitol from glucose was 6.6 percent was obtainedper ml. of a fennentation liquor.

What is claimed is:

l. A process for producing xylitol by fermentation which comprisesinoculating micro-organisms selected from the group consisting ofCandida, Monilia, Pichia, Hansenula, Saccharomyces, Zygosaccharomycesand Debaryomyces which are capable of producing xylitol from D-xylulosein a culture medium containing D-xylulose as a main carbon source,inorganic salts and nitrogen sources and conducting fermentation underaerobic conditions to produce and accumulate xylitol in the culturemedium.

2. A process according to claim 1 wherein type pH of the culture mediumis 6.0.

3. A process according to claim 1 wherein the fermentation is conductedat 30 C.

4. A process according to claim 1 wherein the micro-organisms employedare selected from the group consisting of Candida guilliermondii var.soya ATCC 20,216, Candida lropicalis ATCC 20,215, Monilia vim ATCC20,217, Pichia farinosa ATCC 20,218, Hansenula suaveolens ATCC 20,219,Saccharomyces rouxii ATCC 13356, Zygosaccharomyces gracilis var.italicus ATCC 2623 and Debaryomyces hansenii ATCC 20,220.

5. A process for producing xylitol from glucose which comprises,firstly, inoculating yeasts selected from the group consisting ofCandida, Hansenula, Pichia, Debaryomyces, Saccharomyces, Torulopsis andRhodotorula which are capable of producing D-arabitol from glucose in aculture medium containing glucose as a main carbon source, inorganicsalts and nitrogeneous source, culturing and fennenting under aerobicconditions, thereby producing and accumulating D- arabitol, secondly,adjusting the broth thus obtained to a pH of 6.0, sterilizing the broth,inoculating acetic acid bacteria selected from the group consisting ofAcetobacter suboxydans, and Gluconobacter roseus H 1AM 1840 to thebroth, culturing and fermenting the inoculated broth under aerobicconditions, thereby converting D-arabitol into D-xylulose, and thirdly,adjusting the broth to a pH of 6.0 and sterilizing the broth,inoculating micro-organisms selected from the group consisting ofCandida, Monilia, Pichia, Hansenula, Saccharomyces, Zygosaccharomycesand Debaryomyces capable of producing xylitol from D-xylulose in thesterilized broth, culturing the sterilized broth under aerobicconditions thereby converting the D-xylulose into xylitol.

6. A process according to claim 5 wherein the fermentation is conductedby supplying corn steep liquor in a concentration of 2-8 (w./v.) percentin the above-mentioned third procedure.

7. A process according to claim 5 wherein the concentration of glucoseis 10-30 (w./v.) percent.

2. A process according to claim 1 wherein type pH of the culture mediumis 6.0.
 3. A process according to claim 1 wherein the fermentation isconducted at 30* C.
 4. A process according to claim 1 wherein themicro-organisms employed are selected from the group consisting ofCandida guilliermondii var. soya ATCC 20,216, Candida tropicalis ATCC20, 215, Monilia vini ATCC 20,217, Pichia farinosa ATCC 20,218,Hansenula suaveolens ATCC 20,219, Saccharomyces rouxii ATCC 13356,Zygosaccharomyces gracilis var. italicus ATCC 2623 and Debaryomyceshansenii ATCC 20,220.
 5. A process for producing xylitol from glucosewhich comprises, firstly, inoculating yeasts selected from the groupconsisting of Candida, Hansenula, Pichia, Debaryomyces, Saccharomyces,Torulopsis and Rhodotorula which are capable of producing D-arabitolfrom glucose in a culture medium containing glucose as a main carbonsource, inorganic salts and nitrogeneous source, culturing andfermenting under aerobic conditions, thereby producing and accumUlatingD-arabitol, secondly, adjusting the broth thus obtained to a pH of 6.0,sterilizing the broth, inoculating acetic acid bacteria selected fromthe group consisting of Acetobacter suboxydans, and Gluconobacter roseusH4, IAM 1840 to the broth, culturing and fermenting the inoculated brothunder aerobic conditions, thereby converting D-arabitol into D-xylulose,and thirdly, adjusting the broth to a pH of 6.0 and sterilizing thebroth, inoculating micro-organisms selected from the group consisting ofCandida, Monilia, Pichia, Hansenula, Saccharomyces, Zygosaccharomycesand Debaryomyces capable of producing xylitol from D-xylulose in thesterilized broth, culturing the sterilized broth under aerobicconditions thereby converting the D-xylulose into xylitol.
 6. A processaccording to claim 5 wherein the fermentation is conducted by supplyingcorn steep liquor in a concentration of 2-8 (w./v.) percent in theabove-mentioned third procedure.
 7. A process according to claim 5wherein the concentration of glucose is 10- 30 (w./v.) percent.